Maki Daniels

Immunization delays the onset of prion disease in mice.

The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has been shown to be effective in mouse models of another neurodegenerative condition, namely Alzheimers disease. Here we report that vaccination with recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized before exposure. The increase in the incubation period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a similar approach may work in humans or other mammalian species at risk for prion disease.

High Affinity Binding between Copper and Full-length Prion Protein Identified by Two Different Techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.

A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation.

The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp°/° cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP‐knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.

Astrocytic regulation of NMDA receptor subunit composition modulates the toxicity of prion peptide PrP106-126.

Prion diseases are neurodegenerative conditions. The main pathological alterations common to these diseases include the loss of neurones, gliosis and the deposition of an abnormal isoform of the prion protein in aggregates in the nervous tissue. Prevention of the devastating effects of prion disease requires prevention of neuronal death. Therefore, understanding the mechanism by which this occurs is essential. Cell culture studies using the synthetic peptide PrP106-126 have been central to developing a model of this mechanism. Using a coculture system, we have shown that PrP106-126 caused neuronal death mediated by glutamate. This neuronal death resulted from modification of the expression of NMDA receptor subtypes stimulated by the exposure of neurones to the combination of astrocytic factors, elevated Cu and PrP106-126. The results of these experiments suggest neuronal death in prion disease might be reduced by the use of NMDA receptor antagonists such as MK801 or inhibitors of the arachidonic acid metabolism pathway.

High extracellular potassium protects against the toxicity of cytosine arabinoside but is not required for the survival of cerebellar granule cells in vitro.

Depolarization of cerebellar granule cells with elevated potassium has been described as essential to maintain their survival in culture. There are several reports that this is only specific for rat cerebellar granule cells and not those of mouse. We reinvestigated this issue and found that although high potassium enhanced the survival of cerebellar granule cells from both rat and mouse it was not essential for the survival of those cultures. Further analysis of the culture system indicated that high potassium offered protection against the toxicity of glutamate and cytosine arabinose (Ara C), a standard antimitotic additive to cultures of granule cells. Ara C was found to be toxic to cerebellar cells after potassium withdrawal at concentrations standardly used in culturing these cells (10 microM). High potassium was found to diminish the expression of p53. Ara C toxicity is known to utilize the p53-dependent signaling pathway to initiate apoptosis. Another depolarizing agent, veratridine, offers no protection against Ara C but we provide evidence that the protective effect of high potassium against Ara C is mediated through calcium balance within the cells. We suggest that there is no requirement for high potassium in terms of cerebellar granule cell survival. The previously proposed role for high potassium in the survival cerebellar granule cells is rather a protective effect against toxic substances in serum such as glutamate or against agents such as Ara C.

Negative regulation of Smad2 by PIASy is required for proper Xenopus mesoderm formation.

Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFβ superfamily. Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens. We have identified Xenopus PIASy (XPIASy), a member of the PIAS family, by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait. During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone. XPIASy expression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively. Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation. In addition, their introduction into the ventral marginal zone results in a secondary axis formation. Gain-of-function analysis revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity. These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning.